1. Field of the Invention
The present invention relates generally to the fields of cellular biochemistry and viral replication. More particularly, it concerns the discovery that a certain factor, P-TEFb, has a central role in transcription elongation control, that it phosphorylates RNA polymerase II and that it binds to the HIV protein, Tat. The invention provides human genes encoding the P-TEFb subunits, various other biological components, and methods relating to the control of transcription elongation that have particular utility in the identification of substances that inhibit viral replication.
2. Description of Related Art
The production of any functional eukaryotic mRNA requires efficient transcription elongation by RNA polymerase II. Eukaryotic gene expression is controlled in part during the elongation phase of transcription. Shortly after initiation, RNA polymerase II acquires the properties necessary to synthesize full length pre-mRNAs (Spencer and Groudine, 1990b; Kerppola and Kane, 1991; Wright, 1993; Bentley, 1995; Maldonado and Reinberg, 1995). As is frequently found in control processes, there is a negative control mechanism which is manifest as a blockage during early elongation.
Blocks in transcription, usually referred to as premature termination, have been observed during transcription in a number of systems, including mammalian genes such as α-tubulin (Middleton and Morgan, 1990; Hair and Morgan, 1993), and in viruses, such as adenovirus (Kessler et al., 1989), simian virus 40 (SV40) (Kessler et al., 1991), minute virus of mice (Krauskopf et al., 1991) and human immunodeficiency virus (HIV) (Laspia et al., 1989). RNA polymerase II molecules are also found blocked, during elongation, near the promoter on many genes in Drosophila melanogaster (Rougvie and Lis, 1988; 1990).
Except for the involvement of the viral Tat protein in HIV gene expression (Marciniak and Sharp, 1991), little is known about the molecular mechanisms involved in elimination of this block. In vivo, HIV transcription is tightly controlled by the viral Tat protein. It is known that Tat acts as a potent transcriptional transactivator by binding to the transactivation response (TAR) region on the nascent RNA and interacting with cellular factors (Garcia and Gaynor, 1994; Jones and Peterlin, 1994). Still, many issues remain to be clarified concerning the interactions and functional regulation of transcriptional elongation and Tat, and further information is needed before effective anti-HIV strategies can be developed based upon intervention connected with Tat activity.
Concerning cellular genes, the transcription of the proto-oncogene, c-myc, is regulated by a block during elongation (Miller et al., 1989; Spencer and Groudine, 1990a; Wright and Bishop, 1989). C-myc expression was studied in Xenopus oocytes and isolated HeLa nuclei. The block to elongation in c-myc occurs close to the promoter, termed “promoter-proximal pausing”, and only short RNAs are produced (Krumm et al., 1995; Meulia et al., 1993; Strobl and Eick, 1992). A block to elongation at the end of the first exon regulates the levels of c-fos RNA in response to tumor promoters and intracellular calcium levels (Collart et al., 1991; Mechti et al., 1991). Other blocks to elongation occur in the transcription of the proto-oncogenes c-myb (Bender et al., 1987; Reddy and Reddy, 1989) and c-fins (Yue et al., 1993). The RNA levels for the adenosine deaminase genes (ADA) of humans and mice are at least partly controlled by a regulated block to elongation (Ramamurthy et al., 1990; Chinsky et al., 1989; Chen et al., 1990; Chen et al., 1991; Kash et al., 1993). Thus this elongation control process has been implicated in the expression of many genes, yet the mechanism of control is not yet understood.
Studies in human, murine, Drosophila and Xenopus systems have demonstrated the existence of two classes of elongation complexes differing in their potential to produce full length mRNA sized transcripts. A model for the control of elongation has been described which is based, in part, on results obtained from a Drosophila in vitro transcription system (Kephart et al., 1992; Marshall and Price, 1992) and is consistent with data obtained in vitro and in vivo from many studies.
Key features of the elongation control model are that all RNA polymerase II molecules that initiate from a promoter are destined to produce only short transcripts, in a process termed “abortive elongation”. Abortive elongation is distinct from abortive initiation because the abortive transcripts are 10 to 20 times longer during abortive elongation and, presumably, the polymerase in the abortive elongation complexes must relocate the promoter after producing an abortive transcript to bring about reinitiation. Escape from this negative control is accomplished through the action of P-TEF (positive transcription elongation factor) which allows productive elongation. Fractionation studies have recently identified three components believed to be required to efficiently generate productive elongation complexes, P-TEFa, P-TEFb and factor 2 (Marshall and Price, 1995). P-TEFb was further purified and was shown to act after initiation (Marshall and Price, 1995), although the protein was not subject to detailed biochemical characterization.
The existence of two classes of transcription complexes differing in their elongation potential has also been demonstrated using the nucleoside analog, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). The addition of DRB to mammalian cells in culture resulted in a 95% inhibition in the production of mature mRNA (Sehgal et al., 1976). Nuclei isolated from cells pre-treated with DRB have increased production of short, capped transcripts while labeling of longer RNAs is decreased (Tamm and Kikuchi, 1979; Tamm et al., 1980). Similarly, the short transcripts generated from viral templates in cells infected with SV40 (Laub et al. 1980) and adenovirus (Fraser et al., 1979) are enhanced, while longer transcripts are suppressed with DRB treatment. DRB also inhibits production of long transcripts but leaves shorter products unaffected in injected Xenopus oocytes (Meulia et al., 1993; Roberts and Bentley, 1992).
The carboxyl-terminal domain (CTD) of RNA polymerase II is phosphorylated during the transcription cycle at a time coincident with elongation regulation (Dahmus, 1994; Dahmus, 1995). The CTD can be phosphorylated by the kinase associated with the general transcription factor TFIIH (Lu et al., 1992; Serizawa et al., 1992; Feaver et al., 1991), and a CTD kinase activity is believed to be present in preinitiation complexes at several promoters (Peterson et al., 1992; Kang and Dahmus, 1993). Research has been directed at identifying the CTD kinase, but despite the proposal of various candidate kinases, it appears that the relevant kinase has not yet been identified.
A kinase/cyclin pair (SRB10/11) is part of the holoenzyme form of yeast RNA polymerase II (Liao et al., 1995). A number of other kinases, including casein kinase I and II (Zandomeni et al., 1986; Cadena and Dahmus, 1987), DNA-dependent protein kinase (Dvir et al., 1992), and a murine kinase related to cdc2 and CDC28 (Cisek and Corden, 1989), are capable of phosphorylating the CTD. Also, the kinases CTD-K1 and CTD-K2 purified from HeLa cells (Payne and Dahmus, 1993), CTK1 from yeast (Lee and Greenleaf, 1989), and KI, KII, and KIII from Aspergillus nidulans (Stone and Reinberg, 1992) can all phosphorylate the CTD. It has been further suggested that the stress activated MAP kinases are involved in phosphorylating RNA polymerase II during heat shock (Venetianer et al., 1995). While all of the above are serine/threonine kinases, there is one example of a tyrosine kinase, c-abl, that can phosphorylate the CTD (Baskaran et al., 1993).
While phosphorylation of the CTD has been correlated with the elongation phase of transcription, none of the kinases described above have been shown to modify the functional properties of RNA polymerase II during elongation. Therefore, the identity of the kinase that operates in this control process remains unknown. The mechanism by which CTD phosphorylation induces the transition into productive elongation also remains to be determined, as does the role in elongation control of various proteins associated with RNA polymerase II in a holoenzyme complex (Koleske and Young, 1995). Although several models for the involvement of CTD in elongation control have been proposed (Rasmussen and Lis, 1995), including tethering of the polymerase to the promoter by the unphosphorylated CTD, no conclusive evidence for one model is available. Further work is still needed to determine the fate of polymerases that stop early, but do not enter productive elongation (Marshall and Price, 1992), and to define the interaction of RNA polymerases in early elongation complexes with termination factors, including “factor 2” (Xie and Price, 1996).
Thus, the role of termination factors and potential anti-termination factors, and the regulation mechanisms that operate in the elongation control process remain to be clarified. Not only will the identification of these factors and their respective properties be of significant scientific interest, such discoveries would also have practical values beyond an understanding of transcriptional control mechanisms. For example, many viruses produce viral proteins that somehow interact with RNA polymerase II and facilitate the production of elongated viral transcripts, an essential step in the viral ‘life cycle’. Therefore, the identification and characterization of protein factors involved in productive elongation will likely yield benefits in the development of anti-viral strategies.